The overall goal of this application is to study the molecular and biological role of the NPM-ALK fusion product derived from the translocation 1(2;5) characteristic of anaplastic large cell lymphomas (ALCL). We have also recently demonstrated that in human as well as in murine cells, NPM-ALK activates Jak3, Stat3, and Erk1-2 molecules. More importantly, using conditional Stat3 mouse embryonal fibroblasts (MEF) we have also proven that Stat3 is required for NPM-ALK mediated transformation. Moreover, using NI 7DNRasxNPM-ALK Tg mice we have shown that Ras is necessary for the generation of NPM-ALK T cell lymphomas and for the phosphorylation of serine 727 in Stat3. Nevertheless, the mechanisms leading to the activation of Stat3 and Ras are still elusive and the pathogenetic role of the phosphorylation ofserine 727 in Stat3 remains unclear. In the first Aim, we propose a series of experiments designed to identify the molecular mechanisms leading to the activation of Stat3. We will test whether NPM-ALK is able to directly phosphorylate Stat3 or alternatively if Stat3 requires an adaptor protein which allows it to dock to ALK or to an unknown kinase which in turn will activate Stat3. We also propose to determine the pathogenetic role of Stat3 in NPM-ALK mediated transformation of T lymphocytes. To accomplish this goal we have generated Stat3 conditional T cell specific Tg mice, which were crossed with CD4-NPM-ALK Tg. Because 100 percent of NPM-ALK mice develop lymphomas it is possible to study whether the genetic loss of Stat3 will prevent or delay the occurrence of these neoplasms. Finally, the role of Stat3 in the maintenance of ALK T cell lymphomas will be investigated using FIox/-Stat3/NPM-ALK lymphoma lines after transduction of an inducible Crc retrovirus (CreER-IRES-GFP). Survival of tumor cells and the expression of Bclx and Survivin, genes known to be regulated by Stat3, will be evaluated before and after Stat3 deletion. These studies should not only establish the role of Stat3 in NPM-ALK transformation but more importantly they may reveal more general mechanisms in tumors carrying deregulated Stat3. In the second Aim, we propose to determine the molecular mechanisms leading the Ras activation and to define the role of serine 727 phosphorylation of Stat3 in NPM-ALK mediated transformation. The role of IRS-1, She and Grb2 will be tested using DN and several NPM-ALK mutated constructs. To study the putative tumorigenic role of serine 727, we will take advantage of MEF derived from S727AStat3 knock-in mice. This will be the first genetic approach to dissect the pathogenetic role of the differential phosphorylation status of Stat3. In our third Aim, we will endeavor to prove the requirement of NPM-ALK in the maintenance of NPM-ALK positive lymphomas using a new conditional ploxNPM-ALK Tg. This approach will unequivocally show the role of NPM-ALK in transformed cells and will give us the rationale to test the efficacy of new therapeutic approaches designed to inhibit the function of ALK chimeras.